Digital Microfluidic Devices Research Articles

Abstract A030: Rapid extraction and detection of extracellular vesicle-derived PD-L1 in a microfluidic platform

Abstract Background. Extracellular vesicles (EV) are emerging as new biomarkers for cancer diagnostics and therapeutic monitoring1. For example, in patients that receive PD1/PD-L1 immune checkpoint inhibitors (a key pillar of cancer immunotherapy), circulating EV-derived PD-L1 is gaining attention as a non-invasive biomarker for therapeutic efficacy. Standard methodologies to measure EV-derived PD-L1 requires ultracentrifugation to first separate EVs and then quantify those that express PD-L1 using nano flow cytometry. These are tedious processes that require trained technologists in centralized facilities, which is a key barrier to frequently monitoring EV-based biomarkers. Yet, frequent monitoring is crucial to quickly identify and stop an ineffective therapy while more fruitful options are still available. Method. To bridge the gap, we develop a proof-of-concept digital microfluidic (DMF) device that can separate EVs directly from biofluids and quantify PD-L1+ EVs automatedly. Briefly, DMF device supports automatedly droplet handling, including droplet transport, splitting and mixing, based on electrowetting principles2. Immunomagnetic beads were used to capture EVs from biofluids, and EVs were then detected by electrochemical sensors inserted into the device. These tips of the electrochemical sensors were modified with gold nanoparticle self-assembled layers to enhance target binding, and CD9 antibodies were immobilized on the tips to bind to EVs. We then used anti-PD-L1 as secondary antibodies for target detection. Differential Pulse Voltammetry (DPV)3 were performed to obtain the results. Results. We used the DMF device to extract 109- 1011 #/mL EVs secreted by a human breast cancer cell line (MDA-MB-231). Briefly, 10 mL of cell culture media was loaded into the DMF device, and the sample was mixed and incubated with a 10 mL droplet of immunomagnetic beads to capture EVs. After 5 min, EVs were eluted in 20 mL of elution buffer, and the beads were discarded. We estimated the number of EVs using Nanoparticle Tracking Analysis; the number of EVs extracted on-chip was comparable with those that were extracted using standard in-tube method. We also verified the purity of the EVs extracted on-chip using Western Blot. Our results showed that the electrochemical sensors can detect as low as 104 #/mL PD-L1+ EVs, with a linear range of 104 – 107 #/mL (R2=0.9701). The entire process can be completed within 1 hour. Conclusion. We integrated automated EV extraction and their surface marker detection in a single DMF device. Our next step is to extract and detect EV-based biomarkers from plasma samples. This platform holds promise for the regular monitoring of EV-based biomarkers that are indicators of therapeutic responses in patients that receive cancer immunotherapies.

Automated Droplet Ejection from a Digital Microfluidics Sample Pretreatment Device Enables Batch-Mode Chemiluminescence Immunoassay.

Digital microfluidics (DMF) features programmed manipulation of fluids in multiple steps, making it a valuable tool for sample pretreatment. However, the integration of sample pretreatment with its downstream reaction and detection requires transferring droplets from the DMF device to the outside world. To address this issue, the present study developed a modified DMF device that allows automated droplet ejection out of the chip, facilitated by a tailor-designed interface. A double-layered DMF microchip with an oil-filled medium was flipped over, with a liquid infusion port and a liquid expulsion port accommodated on the top working PCB plate and the bottom grounded ITO plate, respectively, to facilitate chip-to-world delivery of droplets. Using chemiluminescent immunoassay (CLIA) as an illustrative application, the sample pretreatment was programmed on the DMF device, and CLIA droplets were ejected from the chip for signal reading. In our workflow, CLIA droplets can be ejected from the DMF device through the chip-to-world interface, freeing up otherwise occupied electrodes for more sample pretreatment and enabling streamlined droplet microreactions and batch-mode operation for bioanalysis. Integrated with these interfacing portals, the DMF system achieved a single-channel throughput of 17 samples per hour, which can be further upscaled for more productive applications by parallelizing the DMF modules. The results of this study demonstrate that the droplet ejection function that is innovated in a DMF sample pretreatment microsystem can significantly improve analytical throughput, providing an approach to establishing an automated but decentralized biochemical sample preparation workstation for large-scale and continuous bioanalysis.

A digital microfluidic device integrated with electrochemical sensor and 3D matrix for detecting soluble PD-L1

PD1/PD-L1 checkpoint inhibitors are at the forefront of cancer immunotherapies. However, the overall response rate remains only 10–30%. Even among initial responders, drug resistance often occurs, which can lead to prolonged use of a futile therapy in the race with the fatal disease. It would be ideal to closely monitor key indicators of patients’ immune responsiveness, such as circulating PD-L1 levels. Traditional PD-L1 detection methods, such as ELISA, are limited in sensitivity and rely on core lab facilities, preventing their use for the regular monitoring. Electrochemical sensors exist as an attractive candidate for point-of-care tool, yet, streamlining multiple processes in a single platform remains a challenge. To overcome this challenge, this work integrated electrochemical sensor arrays into a digital microfluidic device to combine their distinct merits, so that soluble PD-L1 (sPD-L1) molecules can be rapidly detected in a programmed and automated manner. This new platform featured microscale electrochemical sensor arrays modified with electrically conductive 3D matrix, and can detect as low as 1 pg/mL sPD-L1 with high specificity. The sensors also have desired repeatability and can obtain reproducible results on different days. To demonstrate the functionality of the device to process more complex biofluids, we used the device to detect sPD-L1 molecules secreted by human breast cancer cell line in culture media directly and observed 2X increase in signal compared with control experiment. This novel platform holds promise for the close monitoring of sPD-L1 level in human physiological fluids to evaluate the efficacy of PD-1/PD-L1 immunotherapy.

Investigation into the optoelectrowetting droplet transport mechanism.

To explore the optoelectronic wetting droplet transport mechanism, a transient numerical model of optoelectrowetting (OEW) under the coupling of flow and electric fields is established. The study investigates the impact of externally applied voltage, dielectric constant of the dielectric layer, and interfacial tension between the two phases on the dynamic behavior of droplets during transport. The proposed model employs an improved Young's equation to calculate the instantaneous voltage and contact angle of the droplet on the dielectric layer. Results indicate that, under the influence of OEW, significant variations in the interface contact angle of droplets occur in bright and dark regions, inducing droplet movement. Moreover, the dynamic behavior of droplet transport is closely associated with various parameters, including externally applied voltage, dielectric layer material, and interfacial tension between the two phases, all of which impact the contact angle and, consequently, the transport process. By summarizing the influence patterns of the three key parameters studied, the optimization of droplet transport performance is achieved. The study employs two-dimensional simulation models to emulate the droplet motion under the influence of the electric field, investigating the OEW droplet transport mechanism. The continuous movement of droplets involves three stages: initial wetting, continuous transport, and reaching a steady position. The findings contribute theoretical support for the efficient design of digital microfluidic devices for OEW droplet movement and the selection of key parameters for droplet manipulation.

Polarity-dependent electro-wetting/-dewetting for efficient droplet manipulation

Droplet manipulation on a substrate by electrical signals is instrumental to the automation and miniaturization of labor-intensive assays in life science and chemistry. Current techniques are primarily based on either electrowetting or a more recent ionic-surfactant-mediated electro-dewetting effect. Here, we report that the two effects can occur simultaneously on the same substrate. Using a dope silicon substrate and an aqueous droplet with a cationic surfactant, the surface exhibits dewetting at positive biases and wetting at negative. Such a polarity-dependent wetting–dewetting transition enables a more significant wettability change (>60° contact angle change between ±3 V), which preserves after multiple wetting–dewetting cycles. We also find that the transition does not experience contact angle hysteresis that sole electrowetting commonly suffers from. Benefitting from these features, we experimentally show that droplet manipulation on a digital microfluidic device is more efficient and robust using this joint mechanism.

AIEgens-enhanced rapid sensitive immunofluorescent assay for SARS-CoV-2 with digital microfluidics

BackgroundSensitive and rapid antigen detection is critical for the diagnosis and treatment of infectious diseases, but conventional ELISAs including chemiluminescence-based assays are limited in sensitivity and require many operation steps. Fluorescence immunoassays are fast and convenient but often show limited sensitivity and dynamic range. ResultsTo address the need, an aggregation-induced emission fluorgens (AIEgens) enhanced immunofluorescent assay with beads-based quantification on the digital microfluidic (DMF) platform was developed. Portable DMF devices and chips with small electrodes were fabricated, capable of manipulating droplets within 100 nL and boosting the reaction efficiency. AIEgen nanoparticles (NPs) with high fluorescence and photostability were synthesized to enhance the test sensitivity and detection range. The integration of AIEgen probes, transparent DMF chip design, and the large magnetic beads (10 μm) as capture agents enabled rapid and direct image-taking and signal calculation of the test result. The performance of this platform was demonstrated by point-of-care quantification of SARS-CoV-2 nucleocapsid (N) protein. Within 25 min, a limit of detection of 5.08 pg mL−1 and a limit of quantification of 8.91 pg mL−1 can be achieved using <1 μL sample. The system showed high reproducibility across the wide dynamic range (10–105 pg mL−1), with the coefficient of variance ranging from 2.6% to 9.8%. SignificanceThis rapid, sensitive AIEgens-enhanced immunofluorescent assay on the DMF platform showed simplified reaction steps and improved performance, providing insight into the small-volume point-of-care testing of different biomarkers in research and clinical applications.

Laser-induced graphene-based digital microfluidics (gDMF): a versatile platform with sub-one-dollar cost.

Digital microfluidics (DMF), is an emerging liquid-handling technology, that shows promising potential in various biological and biomedical applications. However, the fabrication of conventional DMF chips is usually complicated, time-consuming, and costly, which seriously limits their widespread applications, especially in the field of point-of-care testing (POCT). Although the paper- or film-based DMF devices can offer an inexpensive and convenient alternative, they still suffer from the planar addressing structure, and thus, limited electrode quantity. To address the above issues, we herein describe the development of a laser-induced graphene (LIG) based digital microfluidics chip (gDMF). It can be easily made (within 10 min, under ambient conditions, without the need of costly materials or cleanroom-based techniques) by a computer-controlled laser scribing process. Moreover, both the planar addressing DMF (pgDMF) and vertical addressing DMF (vgDMF) can be readily achieved, with the latter offering the potential of a higher electrode density. Also, both of them have an impressively low cost of below $1 ($0.85 for pgDMF, $0.59 for vgDMF). Experiments also show that both pgDMF and vgDMF have a comparable performance to conventional DMF devices, with a colorimetric assay performed on vgDMF as proof-of-concept to demonstrate their applicability. Given the simple fabrication, low cost, full function, and the ease of modifying the electrode pattern for various applications, it is reasonably expect that the proposed gDMF may offer an alternative choice as a versatile platform for POCT.

Polar coordinate active-matrix digital microfluidics for high-resolution concentration gradient generation.

Automated concentration gradient generation is one of the most important applications of lab-on-a-chip devices. Digital microfluidics is a unique platform that can effectively achieve digitalized gradient concentration preparation. However, the dynamic range and concentration resolution of the prepared samples heavily rely on the size and the number of effective electrodes. In this work, we report an active-matrix digital microfluidic device with polar coordinate electrode arrangement. The device contains 33 different electrode sizes, generating digital droplets of different volumes. To compare with the conventional rectangular coordinate arrangement with a similar electrode number, this work shows an approximately 19 times resolution enhancement for the achievable concentration gradient. We characterized the stability and uniformity of droplets generated by electrodes of different sizes, and the coefficient of variation of stable droplets was less than 3%. The fluorescent nanomaterial's concentration quantification and glucose concentration characterization experiments were also conducted, and the correlation coefficients for the linearities were all above 0.99.

Dynamic behavior of floating magnetic liquid marbles under steady and pulse-width-modulated magnetic fields.

Liquid marbles show promising potential for digital microfluidic devices due to their lower friction with the platform surface than non-covered droplets. In this study, the manipulation of a biocompatible magnetic liquid marble with a magnetic shell (LMMS) is experimentally studied. The movement of the floating LMMS on the water surface, which is actuated by DC and pulse width modulation (PWM) magnetic fields, is investigated under the influence of various parameters, including the LMMS volume, the initial distance of the LMMS from the magnetic coil tip, the magnetic coil current, the PWM frequency and its duty cycle. The LMMS has a shorter travel time to the magnetic coil tip under a DC magnetic field by increasing the magnetic coil current, decreasing the initial distance and its volume. In the PWM mode, these parameters show similar behavior; moreover, increasing the PWM duty cycle and decreasing the PWM frequency shorten the travel time. It is demonstrated that actuation by a PWM magnetic field with step-by-step movement provides better control over manipulation of the floating magnetic marble. The dynamic behavior of an LMMS is compared to a ferrofluid marble (FM), which is formed using a ferrofluid instead of water as its core. It is observed that the LMMS has a lower velocity than the FM.

Integration of complementary split-ring resonators into digital microfluidics for manipulation and direct sensing of droplet composition.

This paper demonstrates the integration of complementary split-ring resonators (CSSRs) with digital microfluidics (DMF) sample manipulation for passive, on-chip radio-frequency (RF) sensing. Integration is accomplished by having the DMF and the RF-sensing components share the same ground plane: by designing the RF-resonant openings directly into the ground plane of a DMF device, both droplet motion and sensing are achieved, adding a new on-board detection mode for use in DMF. The system was modelled to determine basic features and to balance various factors that need to be optimized to maintain both functionalities (DMF-enabled droplet movement and RF detection) on the same chip. Simulated and experimental results show good agreement. Using a portable measurement setup, the integrated CSSR sensor was used to effectively identify a series of DMF-generated drops of ethanol-water mixtures of different compositions by measuring the resonant frequency of the CSSR. In addition, we show that a binary solvent system (ethanol/water mixtures) results in consistent changes in the measured spectrum in response to changes in concentration, indicating that the sensor can distinguish not only between pure solvents from each other, but also between mixtures of varied compositions. We anticipate that this system can be refined further to enable additional applications and detection modes for DMF systems and other portable sensing platforms alike. This proof-of-principle study demonstrates that the integrated DMF-CSSR sensor provides a new platform for monitoring and characterization of liquids with high sensitivity and low consumption of materials, and opens the way for new and exciting applications of RF sensing in microfluidics.

An integrated and automated digital microfluidic device for dairy milk droplet actuation

An integrated and automated digital microfluidic device for dairy milk droplet actuation

Digital microfluidics-engaged automated enzymatic degradation and synthesis of oligosaccharides.

Glycans are an important group of natural biopolymers, which not only play the role of a major biological energy resource but also as signaling molecules. As a result, structural characterization or sequencing of glycans, as well as targeted synthesis of glycans, is of great interest for understanding their structure-function relationship. However, this generally involves tedious manual operations and high reagent consumptions, which are the main technical bottlenecks retarding the advances of both automatic glycan sequencing and synthesis. Until now, automated enzymatic glycan sequencers or synthesizers are still not available on the market. In this study, to promote the development of automation in glycan sequencing or synthesis, first, programmed degradation and synthesis of glycans catalyzed by enzymes were successfully conducted on a digital microfluidic (DMF) device by using microdroplets as microreactors. In order to develop automatic glycan synthesizers and sequencers, a strategy integrating enzymatic oligosaccharide degradation or synthesis and magnetic manipulation to realize the separation and purification process after enzymatic reactions was designed and performed on DMF. An automatic process for enzymatic degradation of tetra-N-acetyl chitotetraose was achieved. Furthermore, the two-step enzymatic synthesis of lacto-N-tetraose was successfully and efficiently completed on the DMF platform. This work demonstrated here would open the door to further develop automatic enzymatic glycan synthesizers or sequencers based on DMF.

Parylene C as a Multipurpose Material for Electronics and Microfluidics.

Poly(p-xylylene) derivatives, widely known as Parylenes, have been considerably adopted by the scientific community for several applications, ranging from simple passive coatings to active device components. Here, we explore the thermal, structural, and electrical properties of Parylene C, and further present a variety of electronic devices featuring this polymer: transistors, capacitors, and digital microfluidic (DMF) devices. We evaluate transistors produced with Parylene C as a dielectric, substrate, and encapsulation layer, either semitransparent or fully transparent. Such transistors exhibit steep transfer curves and subthreshold slopes of 0.26 V/dec, negligible gate leak currents, and fair mobilities. Furthermore, we characterize MIM (metal-insulator-metal) structures with Parylene C as a dielectric and demonstrate the functionality of the polymer deposited in single and double layers under temperature and AC signal stimuli, mimicking the DMF stimuli. Applying temperature generally leads to a decrease in the capacitance of the dielectric layer, whereas applying an AC signal leads to an increase in said capacitance for double-layered Parylene C only. By applying the two stimuli, the capacitance seems to suffer from a balanced influence of both the separated stimuli. Lastly, we demonstrate that DMF devices with double-layered Parylene C allow for faster droplet motion and enable long nucleic acid amplification reactions.

Digital microfluidic platform assembled into a home-made studio for sample preparation and colorimetric sensing of S-nitrosocysteine.

Digital microfluidics (DMF) is a versatile lab-on-a-chip platform that allows integration with several types of sensors and detection techniques, including colorimetric sensors. Here, we propose, for the first time, the integration of DMF chips into a mini studio containing a 3D-printed holder with previously fixed UV-LEDs to promote sample degradation on the chip surface before a complete analytical procedure involving reagent mixture, colorimetric reaction, and detection through a webcam integrated on the equipment. As a proof-of-concept, the feasibility of the integrated system was successfully through the indirect analysis of S-nitrosocysteine (CySNO) in biological samples. For this purpose, UV-LEDs were explored to perform the photolytic cleavage of CySNO, thus generating nitrite and subproducts directly on DMF chip. Nitrite was then colorimetrically detected based on a modified Griess reaction, in which reagents were prepared through a programable movement of droplets on DMF devices. The assembling and the experimental parameters were optimized, and the proposed integration exhibited a satisfactory correlation with the results acquired using a desktop scanner. Under the optimal experimental conditions, the obtained CySNO degradation to nitrite was 96%. Considering the analytical parameters, the proposed approach revealed linear behavior in the CySNO concentration range between 12.5 and 400μmolL-1 and a limit of detection equal to 2.8μmolL-1. Synthetic serum and human plasma samples were successfully analyzed, and the achieved results did not statistically differ from the data recorded by spectrophotometry at the confidence level of 95%, thus indicating the huge potential of the integration between DMF and mini studio to promote complete analysis of lowmolecular weight compounds.

Asymmetric electrodes for droplet directional actuation by a square wave on an open surface

The large-scale electrowetting-on-dielectric (EWOD) digital microfluidic platforms always require complex electrode-addressing schemes and high-cost fabrication methods of dielectric and hydrophobic layers. Hence, the voltage polarity-dependent electrowetting was used to actuate droplets with asymmetric printed circuit board (PCB) electrodes on an open slippery liquid-infused porous surface (SLIPS) chip. The V-shaped, convex-shaped, and curve-shaped coplanar asymmetric electrode arrays were designed to achieve the directional motion of a droplet by a square wave voltage signal. The operation ranges of voltage amplitude and frequency for continuously actuating a 10 µl droplet were obtained on the three asymmetric electrode arrays. Our results found that the curve-shaped electrode array has a broader voltage operation range than the other two for continuous pumping of a 10 µl droplet. It is because the droplet has a long effective width of contact line with the front curve-shaped electrode and does not overlap with the rear electrode during the movement. Meanwhile, a reconfigurable convex-shaped electrode structure was also proposed to achieve bidirectional actuation of a droplet by controlling the square wave voltage. This work provides a low-cost, straightforward electrode-addressing scheme for designing digital microfluidic devices.

Simple Method to Generate Droplets Spontaneously by a Superhydrophobic Double-Layer Split Nozzle.

Given the problems of traditional droplet generation devices, such as the complex structure and processing technology, difficulty in droplet separation, and low transfer accuracy, we propose a low-adhesion superhydrophobic double-layer split nozzle (SDSN). It realizes spontaneous droplet generation by using an interfacial tension force inside the micro-hole to drive the droplet snap-off. It successfully achieves stable and highly consistent droplets on the micrometer-scale circular micro-hole. Droplets with a volume in the range of 0.65-1.75 ± 0.007 μL can be precisely achieved by adjusting the hole size of the SDSN from 100 to 500 μm. The SDSN is prepared by conventional mechanical drilling, chemical etching, and low surface energy modification. Compared with traditional droplet generation devices, no photolithography process is required, and the cost is lower. Moreover, the droplets can be obtained directly without any post-processing, avoiding the problem of separating droplets from another solution. The stability of SDSN is good, and the droplet volume is not affected by the fluctuation of external conditions. The rate of droplet generation can be freely adjusted by adjusting the speed of the electronic microinjection pump without affecting the droplet volume. It enables efficient droplet transfer without liquid residue, which improves the transfer accuracy and helps to save the use of expensive reagents. This simple but effective structure will be of great help to make breakthroughs in next-generation spontaneous droplet generation, liquid transport, and digital microfluidic devices.

Self-powered digital microfluidics driven by rotational triboelectric nanogenerator

Digital microfluidic (DMF) has emerged as one of the most popular microfluidic platforms for sample-preparation in biochemical analysis and lab-on-a-chip applications. Operated with electrowetting on dielectric (EWOD) mechanism, DMF conventionally requires an external power source to provide the actuation voltage, which limited its portability and broader applications in point-of-care testing (POCT) environment. Herein, a DMF device, self-powered by triboelectric nanogenerator (TENG) is presented. TENG possesses a number of unique characteristics, and is very attractive to be integrated with DMF. It only requires a simple configuration with low-cost fabrication that can improve the DMF portability, but it also provides high voltage, low current output characteristics that are consistent with the EWOD actuation requirements. Basic droplet manipulations, including transportation, split, merge, dispense, and even elongate to follow the electrode patterns of alphabets, on a DMF device powered with manually-rotated Disk-TENG are demonstrated for the first time. Further, droplets containing samples and reagents are transported and mixed on the programmed electrode patterns on the chip to conduct chemical reactions, including nucleic acid amplification and phenol red test, showing that Disk-TENG can serve as the power source for DMF chips in POCT applications.

Digital microfluidics as an emerging tool for bacterial protocols.

Bacteria are widely studied in various research areas, including synthetic biology, sequencing and diagnostic testing. Protocols involving bacteria are often multistep, cumbersome and require access to a long list of instruments to perform experiments. In order to streamline these processes, the fluid handling technique digital microfluidics (DMF) has provided a miniaturized platform to perform various steps of bacterial protocols from sample preparation to analysis. DMF devices can be paired/interfaced with instrumentation such as microscopes, plate readers, and incubators, demonstrating their versatility with existing research tools. Alternatively, DMF instruments can be integrated into all-in-one packages with on-chip magnetic separation for sample preparation, heating/cooling modules to perform assay steps and cameras for absorbance and/or fluorescence measurements. This perspective outlines the beneficial features DMF offers to bacterial protocols, highlights limitations of current work and proposes future directions for this tool's expansion in the field.

Low-cost Polyurethane Coating as Dielectric Component in Digital Microfluidics

Digital microfluidics (DMF) as a platform for precise handling of liquid droplets is a powerful tool but the affordability of the device has been one of the hindrances to its wide implementation. This paper reports the development of DMF devices using low-cost materials and simple deposition techniques specifically for the device dielectric component. Three commercial polyurethane coatings were investigated for their feasibility as the dielectric layer. The electrowetting behaviour of these materials was investigated by evaluating the change in contact angle with applied voltage of a water droplet sitting on the dielectric samples prepared using easy deposition methods such as spraying and spin coating. Devices were then fabricated using these materials to evaluate their capability to actuate droplets. Five types of polyurethane dielectric sample exhibited contact angle reversibility with hysteresis ranging between 4° to 25° after 250 VDC application. Droplet transportation back and forth across 8 electrodes at 180 VRMS has been demonstrated in a device made of one of the polyurethane coatings using a spraying technique. This result implies the potential of using polyurethane for future development of low-cost and disposable DMF devices.

Surface-Enhanced Raman Spectroscopic Probing in Digital Microfluidics through a Microspray Hole.

We report a novel approach for surface-enhanced Raman spectroscopy (SERS) detection in digital microfluidics (DMF). This is made possible by a microspray hole (μSH) that uses an electrostatic spray (ESTAS) for sample transfer from inside the chip to an external SERS substrate. To realize this, a new ESTAS-compatible stationary SERS substrate was developed and characterized for sensitive and reproducible SERS measurements. In a proof-of-concept study, we successfully applied the approach to detect various analyte molecules using the DMF chip and achieved micro-molar detection limits. Moreover, this technique was exemplarily employed to study an organic reaction occurring in the DMF device, providing vibrational spectroscopic data.